Figure 5. The PP-motif is not sufficient to confer the ability to activate TNAP to other ZnT transporters.

(A) Evaluation of the zinc transport activity of hZnT4 mutants using ZnT1^−/− MT^−/− ZnT4 ^−/− cells. ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells stably expressing WT hZnT4 or the hZnT4_MNY-PP mutant were grown in the presence of the indicated concentrations of ZnSO₄ for 72 h, and the number of living cells was measured by the Alamar Blue assay (and plotted as a percentage of living cells at 30 μM ZnSO₄ for each group of cells). ZnT1^−/− MT ^−/− ZnT4 ^−/− cells (●) and ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells stably expressing WT hZnT4 (■) or hZnT4_MNY-PP (▲). Each value is the mean ± S.D. for three independent experiments. (B) TNAP activity in TKO cells is not restored by expressing the hZnT_4MNY-PP mutant. (C) TNAP activity is significantly reduced in TKO cells co-expressing the hZnT5_PP-MNY mutant with hZnT6. In (B) and (C), TNAP activity is expressed as the mean ± S.D. for three independent experiments. The expression of each ZnT protein was confirmed by immunoblotting (lower panels). Tubulin and calnexin (CNX) are shown as loading controls.